The invention concerns an optical sensor and an optical process for the characterization of a chemical and/or bio-chemical substance.
Waveguide grating structures with and without a chemo-sensitive layer are described in the literature (refer to, e.g., EP 0 226 604 B1, EP 0 482 377 A2, PCT WO 95/03538, SPIE Vol. 1141, 192-200, PCT WO 97/09594, Advances in Biosensors 2 (1992), 261-289, U.S. Pat. No. 5,479,260, SPIE Vol. 2836, 221-234).
In EP 0 226 604 B1 and EP 0 482 377 A2, it is demonstrated, how the effective refractive index (resp., the coupling angle) of a chemo-sensitive grating coupler can be measured as a sensor signal. The sensor signal xe2x80x9ceffective refractive indexxe2x80x9d or xe2x80x9ccoupling anglexe2x80x9d is a value which manifests a strong dependence on temperature.
Front-side in-coupling of light into a waveguide (refer to SPIE Vol. 1141, 192-200) is not practical, because a high positioning accuracy is required. In addition, the front side of the wave guide has to be of good optical quality. In PCT WO 95/03538 it is demonstrated how the absolute out-coupling angle of a mode is measured. This value, however, without referencing manifests a high dependence on temperature. In PCT WO 97/09594, chirped waveguide gratings are presented, which, however, also manifest a dependence on temperature.
In Advances in Biosensors 2 (1992), 261-289 it is shown how the disturbing xe2x80x9cpore diffusionxe2x80x9d can be referenced away with the three-layer waveguide model. The refractive index of the waveguiding film manifests drift, while the layer thickness of the waveguiding film (=sensor signal) remains stable. The layout is designed with movable mechanics, which does not permit rapid measurements. In addition, the sensor signal or the light emerging from the waveguide grating structure is recorded from the front side. Front side detection is unsuitable for a two-dimensional layout of waveguide grating structure units. Furthermore, the effective refractive indexes N(TE) and N(TM) for the two polarizations TE and TM are not simultaneously recorded, because for the recording of resonance coupling curves separated by angle a mechanical angular scan is carried out.
In U.S. Pat. No. 5,479,260, a bi-diffractive or multi-diffractive grating coupler is described, whereby the sensor signal is produced by the interferometry of two out-coupling beams of the same or of differing polarization (with the use of a polarizer). Interferometric measurements are complicated, because the intensities of the two beams have to be matched to one another. In addition, temperature fluctuations due to the interferometric signal generated by differing polarizations (using a polarizer) are only partially compensated.
In SPIE Vol. 2836, 221-234, a layout for a waveguide grating structure in connection with fluorescence- or luminescence measurements is described. This layout, however, is not suitable for an (if necessary simultaneous) (absolute) temperature-compensated measurement on the basis of a direct detection. In addition, the waveguide grating structure is mounted on a revolving table.
In Applied Optics 20 (1981), 2280-2283, a temperature-independent optical waveguide is reported about, whereby the substrate is made of silicon. Silicon is absorbent in the visual spectral range. In the case of chemo-sensitive waveguide grating structures, however, the in-coupling takes place in preference from the substrate side. In Applied Optics 20 (1981), 2280-2283, in addition grating couplers which are not temperature-independent are dealt with.
The invention presented here has the object of creating a (bio-)chemo-sensitive optical sensor and to indicate an optical process for the characterization of a (bio-)chemical substance, which do not have the above disadvantages. With the invention, in particular:
(1) sensor signals can be generated, which manifest a low dependence on temperature and/or a low dependence on the diffusion of the specimen liquid into the micropores of a waveguiding film;
(2) both the measurement of (absolute) sensor signals with respect to a direct detection (absolute out-coupling angles xcex1(TE) and xcex1(TM) for the TE- or TM-wave, effective refractive indexes N(TE) and N(TM) for the TE- or TM-wave, layer thickness tF of the waveguiding film etc.) as well as the measurement of (absolute) sensor signals with respect to a marking detection (referenced fluorescence-, luminescence-, phosphorescence signals, etc.), are possible; and
(3) sensor signals remain stable with respect to a slight tilting and/or displacement of the waveguide grating structure, because (local and angular) differences of sensor signals or referenced sensor signals are measured. The object is achieved by the invention as it is defined in the independent claims.
The optical sensor according to the invention contains at least one optical waveguide with a substrate, waveguiding material, a cover medium and at least one waveguide grating structure, at least one light source, by means of which light can be emitted from the substrate side and/or from the cover medium side onto at least a part of the waveguide grating structure, and means for the detection of at least two differing light proportions, whereby with at least one detection agent light emitted into the substrate and/or cover medium can be detected, whereby for the carrying out of a measurement the waveguide can be fixed immovably with respect to the at least one light source and the means of detection.
In the case of the optical process according to the invention for the characterization of a chemical and/or bio-chemical substance in a specimen by means of an optical waveguide containing at least one waveguide grating structure, the specimen is brought into contact with the waveguide in at least one contact zone, in the waveguide in the region of the at least one contact zone at least one light wave is excited through the waveguide grating structure, the at least one light wave is brought into interaction with the specimen, light in at least two differing proportions is detected, of which at least one proportion originates from the region of the contact zone, and at least one absolute measuring signal is generated by the evaluation of the detected light.
The waveguide grating structure consists of one or several waveguide grating structure units, which are arranged one-dimensionally or two-dimensionally (e.g., in a matrix shape or circular shape).
A possible xy-displacement (or only an x-displacement) of the reading head (the reading heads) from one waveguide grating structure to the other or a possible xy-displacement (or only x-displacement) of the waveguide grating structure can quite well be applied.
A waveguide grating structure unit consists of at least two xe2x80x9csensing padsxe2x80x9d (sensor platforms, sensor paths), which differ from one another in at least one of the following characteristics:
(a) The light waves guided in the xe2x80x9csensing padsxe2x80x9d differ in their polarization (TE-wave or TM-wave), whereby the generated sensor signal is not produced by interferometric measurement.
(b) The light waves guided in the xe2x80x9csensing padsxe2x80x9d differ in their mode number.
(c) The two chemo-sensitive layers assigned to the xe2x80x9csensing padsxe2x80x9d manifest a differing specificity (ligand 1 selectively binds (inside or on the surface) to the chemo-sensitive layer covering the xe2x80x9csensing pad 1xe2x80x9d; ligand 2 selectively binds (inside or on the surface) to the chemo-sensitive layer covering the xe2x80x9csensing pad 2xe2x80x9d).
(d) The chemo-sensitive layer assigned to the first xe2x80x9csensing padxe2x80x9d manifests specificity for one ligand (with or without xe2x80x9cnon-specific bindingxe2x80x9d), while the (chemo-sensitive) layer assigned to the second xe2x80x9csensing padxe2x80x9d manifests no specificity (with or without xe2x80x9cnon-specific bindingxe2x80x9d) (example: Dextran layer, to which no identification molecule (e.g., an antibody) is bound).
(e) The light waves guided in the xe2x80x9csensing padsxe2x80x9d differ in their wavelength.
A xe2x80x9csensing padxe2x80x9d, in which guided light waves of differing polriztion (TE-wave or TM-wave) are excited, counts as two xe2x80x9csensing padsxe2x80x9d (difference in the polarization!), providing the sensor signal generated is not produced by interferometric measurement.
A xe2x80x9csensing padxe2x80x9d, in which guided light waves of differing mode number are excited, counts as two xe2x80x9csensing padsxe2x80x9d (difference in the mode number!).
A xe2x80x9csensing padxe2x80x9d, in which guided light waves of differing wavelengths are excited, counts as two xe2x80x9csensing padsxe2x80x9d (difference in the wavelength!).
The first and second xe2x80x9csensing padxe2x80x9d can also be considered as signal- and reference path. The two xe2x80x9csensing padsxe2x80x9d can (but do not have to) have the same structure.
The (bio-)chemo-sensitive layer contacts the waveguiding film in a contact zone. This contact zone normally in the case of the direct detection contains at least one grating. (In the case of interferometric measurements with the same polarization, for a direct detection the (bio-)chemo-sensitive layer can also only be located between two gratings (also refer to EP 0 226 604)). (In the case of interferometric measurements with two differing polarizations (using a polarizer), the (bio-)chemo-sensitive layer can also be located on a multi-diffractive (bi-diffractive) grating (refer to U.S. Pat. No. 5,479,260)).
On principle, for example, the value S(signal path)xe2x88x92cS(reference path) can serve as a possible referenced sensor signal, whereby S(signal path) and S(reference path) are the sensor signals in the first xe2x80x9csensing padxe2x80x9d (signal path) or in the second xe2x80x9csensing padxe2x80x9d (reference path) and c is a calibration factor. In the case of the same polarization, sensibly c=1. In the case of different polarizations, with c the differing sensitivities of the two polarizations can be taken into account. In the case of different wavelengths or mode numbers, with c the differing sensitivities of the wavelengths or of the mode numbers can be taken into account. Advantageously, signal path and reference path are as close together as possible.
The referenced sensor signal in the case of the same polarization furthermore has the advantage that disturbances xcex4, such as, e.g., those caused by temperature fluctuation, light wavelength fluctuation, undesired diffusion of molecules in the waveguides, resp. in the chemo-sensitive layer, unspecific bindings, fluctuations of the concentration of the molecules not to be detected, etc., or combinations of these, can be referenced away, i.e., the referenced sensor signal is independent of xcex4, because S(signal path)+xcex4xe2x88x92(S(reference path)+xcex4)=S(signal path)xe2x88x92S(reference path).
In preference, mono-mode waveguides are utilized, which only carry the fundamental TE-mode or only the fundamental TE-mode and the fundamental TM-mode. The waveguiding film should preferably consist of a high-refractive material, which guarantees the generation of high sensitivities. The waveguiding film can be coated with a chemo-sensitive layer (e.g., an anti-body layer (e.g., suitable for the detection of a corresponding antigen), a dextran layer with identification molecule (e.g., antibodies), receptors, DNA-sections, a silicon layer for the detection of hydrocarbons, etc.). The waveguiding film itself, however, can also represent a chemo-sensitive layer. Rib waveguides can also be utilized.
A xe2x80x9csensing padxe2x80x9d comprises at least one grating, but can also comprise a (possibly more strongly modulated) in-coupling grating and at least one out-coupling grating. The grating periods of in-coupling grating and out-coupling grating can be different (and are in most cases different).
In-coupling grating and out-coupling grating can be uni-diffractive or multi-diffractive grating structures (bi-diffractive gratings, gratings with changing grating period and/or with changing grating diffraction vector, etc.).
A preferred xe2x80x9csensing padxe2x80x9d arrangement consists of three gratings, whereby the middle grating represents the in-coupling grating and the two outer gratings two out-coupling gratings. With a strongly modulated in-coupling grating, for example, one succeeds in exciting modes in forward and reverse direction with a sole (resp., with two) incident (if so required slightly focussed) light beam(s). While a slight displacement of the incident light beam in the mode propagation direction or a slight tilting of the waveguide grating structure with respect to the incident light beam (in the plane of incidence) change the intensity of the modes (running in forward and/or reverse direction) as a result of the changed coupling geometry, not, however, the out-coupling angle(s) or the difference of the out-coupling angles (=double absolute out-coupling angle), which represent possible sensor signals.